1. Field of the Invention
The present invention relates to a method for selective extraction of steroid molecules by entrapped β-cyclodextrin polymers.
2. Description of the Related Art
In general, solid-phase extraction (SPE) via hydrophobic interactions between a packing material and an analyte has been widely used to extract steroid hormones from biological fluids in clinical applications, food analysis, environmental analysis, and doping control for athletes (Journal of Chromatography A, 885: 3-16, 237-250, 321-341, 2000; and Rapid Communications in Mass Spectrometry, 16: 2221-2228, 2002).
In the SPE of steroid hormones using hydrophobic interactions, a modified silica such as C8, C18, phenyl and amino and a copolymer such as XAD (styrene-divinylbenzene copolymer) and Oasis HLB™ (divinylbenzene-N-vinylpypyrrolidone copolymer) are widely used as an adsorbent. However, the silica adsorbent has a drawback, that it is unstable at extreme pH condition during pre-treatment of the sample, becomes deactivated, and the copolymer adsorbent has a drawback that its use is limited in pre-treatment of compounds having high polarity although it is not affected by any pH conditions.
When steroid hormones extracted from biological fluids are analyzed, they are extracted by SPE in pre-treatment and separated from various interfering substances by gas or liquid chromatography combined with mass spectrometry (GC-MS or LC-MS).
Reversed-phase liquid chromatography (RPLC), one of the most widely used techniques to separate steroid hormones from interfering backgrounds in biological samples, also employs hydrophobic interaction (Journal of Chromatography A, 935: 141-172, 2001). Accordingly, biological substances having different chemical properties from the steroids are easily removed by SPE during the pre-treatment process. However, biological substances having similar chemical properties to the steroids are extracted with the steroids and have similar retention time during separation in RPLC.
To overcome the problems, an immuno-affinity chromatography based on biological specificity has been used (Journal of Chromatography A, 794: 37-43, 1998; Rapid Communications in Mass Spectrometry, 16: 370-374, 2002); however, it is limited because of time-consuming process to obtain antibodies of steroid hormones and the high cost of the process.
Meanwhile, β-cyclodextrin, unlike the conventional adsorbents, is capable of sequestering an organic compound having a particular structure or a steroid hormone within its inner cavity (Journal of Separation Science, 25: 789-813, 2002; Steroids, 68: 321-327, 2003).
Korean Patent Application Nos. 1997-0018599, 1997-0037127 and 1997-0037128 disclose methods of removing cholesterol, a starting substance to make steroid hormones from a liquid such as milk and cream using the property of β-cyclodextrin. However, the methods have a drawback that a small amount of β-cyclodextrin still remains even after centrifugation in the process of separating the β-cyclodextrin, to which cholesterol is adsorbed, from the liquid such as milk and cream from the liquid.
To solve the above problem, there was introduced a method to remove cholesterol having a structure similar to that of steroid hormones from milk by chemically immobilizing β-cyclodextrin onto glass beads as a SPE (Archives of Pharmaceutical Research, 27: 873-877, 2004). However, this method is not efficient because the activity of β-cyclodextrin, which is chemically immobilized onto the glass beads, is reduced due to chemical treatments and thus cholesterol cannot be removed efficiently.
In addition, there was introduced a method to increase cholesterol removal rate by forming cross-links among β-cyclodextrin molecules using epichlorohydrin as a chemical linker in an aqueous NaOH solution to increase β-cyclodextrin activity (Archives of Pharmaceutical Research, 27: 1183-1187, 2004). Various polymerized β-cyclodextrins have been prepared under varied conditions during the cross-linking of β-cyclodextrin molecules and physical properties and activities of the β-cyclodextrin have been measured (Polymer International, 54: 744-753, 2005). However, the polymerized β-cyclodextrin prepared using the epichlorohydrin forms a gel state which becomes swollen when used in extracting steroid hormones from biological fluids, such as urine and plasma. Thus, it gives a problem to use only a very small amount of the polymerized β-cyclodextrin in the pre-treatment relative to the amount of the sample.